Biological control of dipteran pests of the genus Lycoriella using Bacillus thuringiensis

ABSTRACT

The invention relates to the biological control of Dipteran pests other than the mosquito, which comprises the use of Bacillus thuringiensis isolate HD 541 (NCIMB 40373) or HD 571 (NCIMB 40374), or a mutant, recombinant or derivative thereof, or insecticidal material derived therefrom.

This invention relates to biological methods of pest control and moreparticularly to methods and compositions utilizing Bacillusthuringiensis for the control of agricultural, horticultural and publichealth pests.

Bacillus thuringiensis is a spore forming bacterium which has long beenknown to kill insects. Many hundreds of isolates (strains) of thisspecies have been listed. Cultures of these isolates are catalogued andmaintained by the Agricultural Research Services of the US Department ofAgriculture, Peoria, Ill., and a number of other internationalcollections, and are available to researchers on request. The release ofsome cultures require the permission of the contributor to the culturecollection but most are available without any conditions. The spectrumof activity of these isolates and their potency against particularinsects vary very widely.

The present invention comprises the use of B. thuringiensis isolatesHD541 and HD571 for the control of Dipteran pests which affect plantsand edible fungi. Accordingly the invention comprises a method of pestcontrol in which agricultural and horticultural produce are protectedagainst pests by the application of isolates of the sub-specieskyushuensis to the growing plants or other horticultural products or totheir growing locations. The invention also comprises compositionscontaining one or more B. thuringiensis isolates formulated foragricultural, horticultural or public health application. The twoisolates, listed as HD 541 and HD 571 in the US culture collectionmentioned above, have shown excellent promise for the purposes of thepresent invention. These have been available at the US culturecollection since August 1979 (HD541) and December 1979 (HD571) and areavailable without restriction. However, in order to ensure theircontinued availability we have deposited them on Mar. 8th 1991 with theNational Collection of Industrial and Marine Bacteria, 23 St. MacharDrive, Aberdeen AB2 IRY, Scotland under the Budapest Treaty. Thedeposited strains have been given Accession numbers NCIMB 40373 (HD541)and NCIMB 40374 (HD571).

The B. thuringiensis isolates HD541 and HD571 are known to have aneffect on the dipteran insect Aedes aegypti (mosquito). However thelevel of activity against this insect is low, being approximately tenthousand-fold less than that of B. thuringiensis israelensis, thesub-species which is currently used for mosquito control. Isolates HD541and HD571 have also been tested against other mosquito species but theirpotency was found to be too low to be useful for practical purposes.

It has now been found that the named isolates are highly potentinsecticides for certain Dipteran pests other than the mosquito,especially for plant and mushroom pests such as the sciarid fungusgnats. The isolates of the invention, i.e. HD541 and HD571 are highlyactive against larvae of the mushroom sciarid Lycoriella auripila, themost damaging pest of mushroom crops in the UK, which causes severelosses in crop yield and is at present controlled mainly by the chemicalinsecticide diflubenzuron. A similar species, L. mali, occupies the sameniche in the USA and Australia. The activity of the named isolatesagainst these pests is significantly greater than that of other isolatesof B. thuringiensis including some from each of the sub-speciesisraelensis, dendrolimus, kyushuensis and morrisoni. The named isolatesthus provide much needed new agents for the control of these pests. Inaddition to the sciarids, other mushroom pests which may be controlledby these isolates are the mushroom phorids. However, the named isolatesdo not have unusually good activity against mushroom cecid pests. OtherDipteran pests of agricultural significance include the Mediterraneanfruit fly.

As indicated, the present invention utilises the natural isolates HD541and HD571. The invention is applicable also to mutants, recombinants andderivatives of the natural isolates and which are active againstDipteran pests. Such derivatives include those obtained by geneticmanipulation. The invention also includes the use of delta endotoxinsderived from the above-mentioned organisms.

The invention provides an entomocidal substance derived from Bacillusthuringiensis strain HD541 or HD571, or from a derivative or mutantthereof. In one embodiment the entomocidal substance is a spore-crystalcomplex.

The invention further provides an entomocidal substance derived fromBacillus thuringiensis strain HD541 (NCIMB 40373) or HD571 (NCIMB 40374) or a derivative or mutant thereof, or an entomocidal substance asdefined above together with an agricultural adjuvant such as a carrier,diluent, surfactant or application-promoting adjuvant. The compositionmay also contain a further biologically active compound selected fromfertilizers, micronutrient donors, plant growth preparations,herbicides, insecticides, fungicides, bactericides, nematicides andmolluscicides and mixtures thereof. The composition may comprise from0.1 to 99% by weight of Bacillus thuringiensis HD541 or HD571 or thederivative or mutant thereof, or the entomocidal substance; from 1 to99.9% by weight of a solid or liquid adjuvant and from 0 to 25% byweight of a surfactant.

The invention in addition provides a method of combatting pests whichcomprises applying to the pests or to their environment an entomocidallyeffective amount of Bacillus thuringiensis strain HD541 or HD571 , or aderivative or mutant thereof, or an entomocidal substance as definedabove, or a composition containing said strain, derivative, mutant orsubstance.

The strains of Bacillus thuringiensis HD541 or HD571 or the compositionscontaining them may be administered to the plants or crops to beprotected together with other insecticides or chemicals without loss ofpotency.

It is compatible with most other commonly used agricultural spraymaterials but should not be used in extremely alkaline spray solutions.

It may be administered as a dust, a suspension, a wettable powder or inany other material form suitable for agricultural application.

During production by fermentation, after normal growth of Bacillusthuringiensis, the mother cells lyse and release the spores and crystalsinto the growth medium. The spores and crystals may be harvested bycentrifugation or filtration, spray drying, vacuum drying, or a methodof precipitation, such as the lactose coprecipitation technique asreported by Dulmage et al (Journal of Invertebrate Pathology, 15, 15-20,1970). The resulting spore-crystal complex is stable for long periodsand can be formulated into a product suitable for application to crops.

A method for preparing an insecticidal composition according to theinvention, comprises culturing the Bacillus thuringiensis strain HD541or HD571 by:

a) maintaining the strain in lyophilized ampules,

b) inoculating with the strain on agar slopes,

c) incubating these slopes for 1 to 5 days at 20° to 40° C., preferably25° to 33° C.

d) inoculating from these slopes into shaken flasks containing anaqueous culture medium,

e) shaking this container at a temperature of 20° to 40° C. preferably30° C. for 1 to 5, preferably 1 to 2 days and optionally repeating thisvegetative growth stage at least once in a separate flask,

f) inoculating in a preculture fermenter an aqueous cultivating mediumwith the cultures of stage e),

g) stirring and aerating the medium containing the inoculate at atemperature of 20° to 40° C., preferably 30° to 35° C., and optionallyrepeating this preculture fermentation stage at least once in a separatelarger container,

h) introducing 2 to 20 per cent by weight of the incubating liquor ofstage g) into a production fermenter, containing an aqueous cultivatingmedium,

i) stirring and aerating the medium at a temperature of 20° to 40° C.,preferably 30° to 35° C.

j) harvesting the Bacillus thuringiensis HD541 or HD571 broth whensporulation and crystal production in the production fermenter reaches amaximum.

k) the agar and broth in a to d should contain at least one nitrogensource, at least one carbon source and at least one salt, preferablypeptone, glucose and at least one salt. The media in f to j shouldcontain at least one nitrogen source (eg peptone, yeast extract, cornsteep liquor, soya bean meal, cotton seed meal, fishmeal ), at least onecarbohydrate source (eg glucose, lactose, sucrose, starch or rawmaterial rich in these constituents) and at least one mineral salt. Thenitrogen and carbohydrate should be balanced to exhaust as near aspossible simultaneously.

The spore-crystal complex or the composition containing it may beadministered to the plants or crops to be protected together withcertain other insecticides or chemicals without loss or potency.

It is possible to kill the spores in the spore-crystal (eg by gammairradiation), or to avoid producing spores by use of an asporogenouscrystali ferous mutant, thereby producing a non-viable product. Anon-viable product may be advantageous in certain circumstances where itis desired to prevent the spread of bacteria for aesthetic reasons or toavoid causing disease in beneficial insects. However, non-viableproducts are generally not as active as those containing live spores andas a further disadvantage there is the increased cost of killing thespores.

The invention furthermore relates to a method of treating crops whichcomprises applying an entomocidally effective amount of B. thuringiensisHD541 or HD571, or a composition thereof.

Bacillus thuringiensis HD541 or HD571, is normally applied in the formof compositions and can be applied to the crop area to be treated,simultaneously or in succession, with further biologically activecompounds. These compounds may be both fertilizers or micronutrientdonors or other preparations that influence plant growth. They may alsobe selective herbicides, insecticides, fungicides, bactericides,nematicides, molluscicides or mixtures of several of these preparations,if desired together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, eg natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders or fertilizers.

The formulations, ie the compositions, preparations or mixturescontaining B. thuringiensis HD541 or HD571 as an active ingredient orcombinations thereof with other active ingredients, and, whereappropriate, a solid or liquid adjuvant, are prepared in known manner egby homogeneously mixing and/or grinding the active ingredients withextenders eg solvents, solid carriers and in some cases surface-actingcompounds (surfactants).

Suitable solvents are: aromatic hydrocarbons, preferably the fractionscontaining 8 to 12 carbon atoms, eg xylene mixtures or substitutednapthalenes, phthalates such as dibutylphthalate or dioctylphthalate,aliphatic hydrocarbons such as cyclohexane or paraffins, alcohols andglycols, such as cyclohexanone, strongly polar solvents such asN-methyl-2-pyrrolidone, dimethylsulfoxide or dimethylformamide as wellas vegetable oils or epoxidized vegetable oils such as epoxidizedcoconut oil or soybean oil; or water.

The solid carriers used eg for dusts and dispersable powders, arenormally natural mineral fillers such as calcite, talcum, kaolin,montmorillonite or attapulgite. In order to improve the physicalproperties of the carriers it is possible to add highly dispersedsilicic acid or highly dispersed absorbent polymers. Suitable granulatedadsorptive carriers are porous types, for example pumice, broken brick,sepiolite or bentonire; and suitable nonsorbent carriers are materialssuch as calcite or sand. In addition, a great number of pregranulatedmaterials of inorganic or organic nature can be used, eg especiallydolomite or pulverised plant residues.

Depending on the nature of the active ingredients to be formulated,suitable surface-active compounds are non-ionic, cationic and/or anionicsurfactants having good emulsifying, dispersing and wetting properties.The term "surfactants" will also be understood as comprising mixtures ofsurfactants.

Suitable anionic surfactants can be both water-soluble soaps andwater-soluble synthetic surface-active compounds.

Suitable soaps are the alkali metal salts, alkaline earth metal salts orunsubstituted or substituted ammonium salts of higher fatty acids (C₁₀-C₂₂), eg the sodium or potassium salts of oleic or stearic acid, or ofnatural fatty acid mixtures which can be obtained, eg from coconut oilor tallow oil. Further suitable surfactants are also the fatty acidmethyltaurin salts as well as modified and unmodified phospholipids.

More frequently, however, so-called synthetic surfactants are used,especially fatty sulfonates, fatty sulfates, sulfonated benzimidazolederivatives or alkylaryl-sulfonates.

The fatty sulfonates or sulfates are usually in the forms of alkalimetal salts, alkaline earth metal salts or unsubstituted or substitutedammonium salts and generally contain a C₈ -C₂₂ alkyl radical which alsoincludes the alkyl moiety of acyl radicals, eg the sodium or calciumsalt of lignosulfonic acid, or dodecylsulfate, or of a mixture of fattyalcohol sulfates obtained from natural fatty acids. These compounds alsocomprise the salts of sulfuric acid esters and sulfonic acids of fattyalcohol/ethylene oxide adducts. The sulfonated benzimidazole derivativespreferably contain 2 sulfonic acid groups and one fatty acid radicalcontaining about 8 to 22 carbon atoms. Examples of alkyl-arylsufonatesare the sodium, calcium or triethanolamine salts ofdodecylbenzenesulfonic acid, dibutylnapththalenesulfonic acid or of anaphthalenesulfonic acid/formaldehyde condensation product. Alsosuitable are corresponding phosphates, eg salts of the phosphoric acidester of an adduct of p-nonylphenol with 4 to 14 moles of ethyleneoxide.

Non-ionic surfactants are preferably polyglycol ether derivatives ofaliphatic or cycloaliphatic alcohols or saturated or unsaturated fattyacids and alkylphenols, said derivatives containing 3 to 30 glycol ethergroups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon moietyand 6 to 18 carbon atoms in the alkyl moiety of the alkylphenols.

Further suitable non-ionic surfactants are the water-soluble adducts ofpolyethylene oxide with polypropylene glycol,ethylenediaminopolypropylene glycol and alkylpolypropylene glycolcontaining 1 to 10 carbon atoms in the alkyl chain, which adductscontain 20 to 250 ethylene glycol ether groups and 10 to 100 propyleneglycol ether groups. These compounds usually contain 1 to 5 ethyleneglycol units per propylene glycol unit.

Representative examples of non-ionic surfactants arenonylphenolpolyethoxyethanols, castor oil polyglycol ethers,polypropylene/polyethylene oxide adducts, tributylphenoxypolyethoxyethanol, polyethylene glycol and octylphenoxypol yethoxyethanol.Fatty acid esters of polyoxyethylene sorbitan, such as polyoxyethylenesorbitan trioleate, are also suitable non-ionic surfactants.

Cationic surfactants are preferably quaternary ammonium salts whichcontain, as N-substituent, at least one C₈ -C₂₂ alkyl radical and, asfurther substituents, lower unsubstituted or halogenated alkyl, benzylor hydroxyl-lower alkyl radicals. The salts are preferably in the formof halides, methylsulfates or ethyl sulfates eg stearyltrimethylammoniumchloride or benzyldi-(2-chloroethyl ) ethylammonium bromide.

The surfactants customarily employed in the art of formulation aredescribed eg in "McCutcheon's Detergents and Emulsifiers Annual", MCPublishing Corp, Ridgewood, N.J., 1979; Dr Helmut Stache, "TensidTaschenbuch" (Handbook of Surfactants ), Carl Hanser Verlag,Munich/Vienna.

The entomocidal compositions usually contain 0.1 to 99%, preferably 0.1to 95% of Bacillus thuringiensis HD541 or HD571, or combination thereofwith other active ingredients, 1 to 99.9% of a solid or liquid adjuvantand 0 to 25%, preferably 0.1 to 20%, of a surfactant.

Whereas commercial products are preferably formulated as concentrates,the end user will normally employ dilute formulations of substantiallylower concentration.

The compositions may also contain further ingredients, such asstabilizers, antifoams, viscosity regulators, binders, tackifiers aswell as fertilizers or other active ingredients in order to obtainspecial effects.

The invention will now be further described with reference toexperimental and field test data obtained in comparative testing ofisolates HD541 and HD571 and of other B. thuringiensis isolates.

EXAMPLE 1--Bacterial Culture

Bacteria were grown at 30° C. in 50 ml of a growth medium, contained in250 ml fluted flask on an orbital shaker at 250 rpm and inoculated at arate of 1% v/v with a mid-exponential phase culture. A typical growthmedium comprised 10 g/l soymeal, 10 g/l potato starch, 5 g/l K₂ HPO₄ and10 ml/l trace elements solution. The trace elements solution comprised 8g/l CaCl₂. 2H₂ O, 5 g/l MnCl₂. 2H2O and 0.5 g/l of each of CuCl₂. 2H₂ O,ZnCl₂, CoCl₂ and FeCl₃. Cultures were monitored by phase-contrastmicroscopy and harvested upon complete autolysis of the cells. Durationof incubation ranged from 40 to 72h, depending on the isolate.Preparation of the growth medium and conversion of cultures into drypowders by the lactose-acetone method were as described by Dulmage et al(Journal of Invertebrate Pathology 1970, 15, 15-20). Production ofbacteria for mushroom trials was the same as above except that 400ml ofbroth was used in 2 l fluted flasks and the broth was not made into apowder.

EXAMPLE 2--Insect Bioassays

For the laboratory bioassay, bacterial powders were homogenized inde-ionized water, diluted as appropriate and 10 ml volumes mixed into 40g of a peat: soya-flour mixture (20:1). The treated medium was thenequally split into two plastic pots (A. W. Gregory Ltd, London) and aminimum of 20 neonate Lycoriella auripila or L. mali larvae added perpot. Eggs and larvae of L. auripila or L. mali were obtained using themethod devised by Binns (Annals of Applied Biology 1973, 73, 119-126).Pots were incubated at 24° C. and subsequent emergence of adultsrecorded daily. Emergence of adults from eight control pots without B.thuringiensis was used for treatment comparisons.

All isolates were initially tested at the rate of 10 mg of bacterialpowder per g of medium. Those strains which caused greater than 95%mortality were re-tested in greater detail using a range ofconcentrations. All data were analyzed by Probit analysis.

Activity of the B. thuringiensis isolates against L. auripila variedconsiderably, with 9 out of 22 isolates having good to moderate activity(Table 1). Strains belonging to sub-species morrisoni, darmstadiensis,kyushuensis, and israelensis were the most active. Flies emerging fromtreatments containing sublethal doses of B. thuringiensis also tooklonger to develop through to the fly stage than did the controls.Development times were, on average, 4 days longer in those treatmentswhich caused greater than 50% mortality in laboratory assays.Statistical analysis of the bioassay data showed that isolates HD541 andHD571 were significantly more active than any of the others rested.

In separate tests, isolates HD541 and HD571 were found to besignificantly more active against L. auripila than other isolatesbelonging to sub-species kyushuensis.

A comparison was made of the activity of gamma-irradiated samples ofisolates HD571 and IPS82 against L. mali. B. thuringiensis isolate HD571was found to be significantly more active (LC₅₀ 44.4 ug per g, fiduciallimits 27.40-58.75) than B thuringiensis israelensis isolate IPS82,which had an LC₅₀ value in excess of 100 ug per g.

EXAMPLE 3--Mushroom Trials

Spawn-run mushroom compost, was put into 48, 25 cm plastic plant pots ata rate of 2.5 kg per pot. A moist casing mixture, consisting of equalvolumes of peat and chalk, was added to the surface of the compost at arate of 1.5 kg per pot. Previous to its application, the casing waseither left untreated; treated with diflubenzuron at 30 mg a. i./kg--the standard commercial control chemical and rate; or treated withbroth cultures or formulations of the strains at four rates: 0.4, 2, 10,or 50 ml/kg of casing for HD 541 and 4.0, 10.0, 25.0 or 62.5 ml/kg ofcasing for HD 571. The six treatments were each replicated eight times.

Six pots, one from each treatment, were arranged at random in a woodenmushroom tray (0.9×0.6m) and placed into a mushroom chamber. There were8 trays in all, arranged into two stacks of 4. The chamber contained ahigh population of L. auripila adults. After 7 days the adults werekilled with an aerial application of synthetic pyrethroids to preventfurther egg laying. Eleven days after treatment, sticky traps wereplaced on top of the casing to catch emerging adults. The pots were thenindividually enclosed in fly-proof polyester netting. The traps wereexamined for flies 18, 22, 28 and 33 days after treatment. The airtemperature within the chamber was maintained at 20° C. for the durationof the experiment.

Addition of broth cultures of the isolates HD541 and HD571 to the casinglayer in the mushroom trials resulted in significant reduction insubsequent emergence of L. auripila adults compared to the untreatedcontrol (Table 2 (a)+(b)). HD 541 at 50 ml/kg and HD 571 atconcentrations above 10 ml/kg gave comparable levels of control to thatof the diflubenzuron treatment.

EXAMPLE 4

Formulation Examples for solid active ingredients of Bacillusthuringiensis HD541 or HD571 or combinations thereof with other activeingredients

(throughout, percentages are by weight)

    ______________________________________    1. Wettable powders  a)      b)      c)    ______________________________________    Bacillus thuringiensis HD541 or HD571                         25%     50%     75%    sodium lignosulfonate                         5%       5%     --    sodium laurylsulfate 3%      --       5%    sodium diisobutylnapthalenesulfonate                         --       6%     10%    octylphenol polyethylene glycol ether    (7-8 moles of ethylene oxide)                         --       2%     --    highly dispersed silicic acid                         5%      10%     10%    kaolin               62%     27%     --    ______________________________________

Bacillus thuringiensis HD541 or HD571, is thoroughly mixed with theadjuvants and the mixture is thoroughly ground in a suitable mill,affording wettable powders which can be diluted with water to givesuspensions of the desired concentration.

    ______________________________________    2. Emulsifiable concentrate    ______________________________________    Bacillus thuringiensis HD541 or HD571                                  10%    octylphenol polyethylene glycol ether (4-5 moles of ethyl-                                   3%    ene oxide)    calcium dodecylbenzenesulfonate                                   3%    castor oil polyglycol ether (36 moles of ethylene oxide)                                   4%    cyclohexanone                 30%    xylene mixture                50%    ______________________________________

Emulsions of any required concentration can be obtained from thisconcentrate by dilution with water.

    ______________________________________    3. Dusts                a)     b)    ______________________________________    Bacillus thuringiensis HD541 or HD571                             5%     8%    talcum                  95%    --    kaolin                  --     92%    ______________________________________

Ready for use dusts are obtained by mixing the active ingredient withthe carriers and grinding the mixture in a suitable mill.

    ______________________________________    4. Extruder granulate    ______________________________________    Bacillus thuringiensis HD541 or HD571                               10%    sodium lignosulfonate       2%    carboxymethylcellulose      1%    kaolin                     87%    ______________________________________

The active ingredient or combination is mixed and ground with theadjuvants and the mixture is subsequently moistened with water. Themixture is extruded, granulated and then dried in a stream of air.

    ______________________________________    5. Coated granulate    ______________________________________    Bacillus thuringiensis HD541 or HD571                                3%    polyethylene glycol (mol. wt. 200 daltons)                                3%    kaolin                     94%    ______________________________________

The finely ground active ingredient or combination is uniformly appliedin a mixter to the kaolin moistened with polyethylene glycol. Non-dustycoated granulates are obtained in this manner.

    ______________________________________    6. Suspension Concentrate    ______________________________________    Bacillus thuringiensis HD541 or HD571                                   40%    ethylene glycol                10%    nonylphenol polyethylene glycol ether (15 moles of ethyl-                                    6%    ene oxide)    sodium lignosulfonate          10%    carboxymethylcellulose          1%    37% aqueous formaldehyde solution                                  0.2%    silicone oil in the form of a 75% aqueous emulsion                                  0.8%    water                          32%    ______________________________________

The finely ground active ingredient or combination is intimately mixedwith the adjuvants giving a suspension concentrate from whichsuspensions of any desired concentration can be obtained by dilutionwith water.

EXAMPLE 5--Formulations: Efficacy Tests

A number of formulations of isolate HD571 were prepared as described inExample 4 and compared with unformulated culture broth and unformulatedspray dried broth for control of L. auripila in a mushroom trial. Theresults, expressed as a percentage of pest control observed indiflubenzuron-treated pots, are summarized in Table 3. All preparationsof isolate HD571 exhibit activity against L. auripila although the levelof activity varies with the type of formulation. These representexamples of only a few effective formulations, preparation of many othereffective formulations will be known to those skilled in the art.

                  TABLE 1    ______________________________________    Activity of Bacillus thuringiensis against Lycoriella auripila.                        LC.sub.50    Code    Sub-species ug Et/g medium                                     Fiducial limits    ______________________________________    IPS-82  israelensis 130.4        95.9-180.0    HD657   "           154.4        103.4-223.8    HD 240  galleriae   2027.6       998.6-4530.6    PG 14   morrisoni   117.9        88.3-156.9    HD 753  darmstadiensis                        144.0        84.9-216.5    HD 754  "           330.2        225.6-537.5    HD 573  wuhanensis  2304.9       1575.4-3064.0    HD 541  kyushuensis 31.4         20.9-42.3    HD 571  "           12.5         9.2-16.9    ______________________________________

Thirteen isolates involving 11 other sub-species had little or noactivity (0 to 77% mortality at the very high dose of 10,000 ug of B.thuringiensis powder/g medium).

                  TABLE 2    ______________________________________    The effect of two strains of Bacillus thuringiensis and    diflubenzuron on the total number of Lycoriella auripila flies    emerging from treated casing.    (a) Strain HD541    Treatment             HD541               Dif*   Control    ______________________________________    Rate ˜             0.4     2.0     10.0  50.0  30.0 --    Total No 1602    1301    901   192   193  2071    % of Control             77.4    62.8    43.5  9.3   9.3  100.0    ______________________________________    (b) Strain HD571    Treatment             HD571               Dif*   Control    ______________________________________    Rate ˜             4.0     10.0    25.0  62.5  30.0 --    Total No 313     81      31    10    22   754    % of Control             41.5    10.7    4.1   1.3   2.9  100.0    ______________________________________     *Diflubenzuron formulated as a 25% wettable powder     ˜HD 571 & HD 541, mls broth/kg casing; Diflubenzuron, mg a.i./kg

                  TABLE 3    ______________________________________    Activity of B. thuringiensis HD571 formulations against    Lycoriella auripila    Formulation        Activity.sup.1    ______________________________________    Culture broth      100    Spray-dried culture broth                       85    WP 50.sup.2        80    SC 100.sup.3       85    SC 200.sup.4       65    ______________________________________     .sup.1 Activity expressed as % of control achieved by diflubenzuron.     Formulations were added to casing at 0.625 g per kg (or equivalent).     .sup.2 Wettable powder containing 50% B. thuringiensis HD571 and prepared     as described in Example 4.     .sup.3 Suspension concentrate (diluted 10 fold) prepared as described in     Example 4.     .sup.4 Suspension concentrate (diluted 5 fold) prepared as described in     Example 4.

We claim:
 1. A method for the biological control of Dipteran pests ofthe genus Lycoriella, comprising contacting the pests with a Lycoriellacontrolling effective amount of a strain of Bacillus thuringiensissub-species kyushuensis selected from group consisting of Bacillusthuringiensis HD541 (NCIMB 40373), Bacillus thuringiensis HD571 (NCIMB40374) and mutants thereof having the same insecticidal activity.
 2. Themethod according to claim 1 which the Dipteran pests of the genusLycoriella are those which affect plants and edible fungi.
 3. The methodaccording to claim 1 wherein the Dipteran pests of the genus Lycoriellaare mushroom pests.
 4. The method according to claim 1 wherein theDipteran pests of the genus Lycoriella are Lycoriella auripila orLycoriella mali.